Application of pulsed focused ultrasound (pFUS) to deliver engineered therapeutic exosomes for the treatment of ischemic stroke

We have shown enhanced delivery of endothelial progenitor cell (EPC) derived naïve exosomes to the sites of stroke using pulsed-focused ultrasound (pFUS) without causing damage to the surrounding brain tissues. Preliminary results from the study also showed that enhanced delivery of EPC exosomes (by pFUS) caused significantly decreased stroke volume and improved cerebral blood flow over 28 days compared to that of EPC exosomes only treated animals. Therefore, pFUS without nanobubbles can be used as a method to enhance the delivery of IV-administered exosomes at the site stroke. We have developed a platform to make engineered exosomes using non-tumorous HEK293 cells that carry and express specific cell-targeting peptides as well as therapeutic probes. The objectives of this proposal are to develop engineered exosomes using our platform to carry neuron-specific rabies virus glycoprotein (RVG) peptides as targeting ligands for the central nervous system and to carry a targeted therapeutic probe, neuroglobin (Ngb), to protect neurons from hypoxic/ischemic and oxidative stress-related insults following ischemic stroke. We hypothesize that engineered exosomes carrying both targeting RVG peptides and therapeutic Ngb protein can be delivered to the site of ischemic stroke and the delivery of these exosomes will be enhanced using pFUS to improve the benefits of post-stroke exosome therapy. The hypothesis will be tested with the following specific aims: Specific Aim 1: Investigate the characteristics of the engineered exosomes and in vivo biodistribution of intravenously (IV) administered exosomes in stroke areas with or without pFUS. Characteristics of the engineered exosomes will be investigated to determine the size, zeta potential, and the presence of peptide and protein on the surface or inside the lumen using techniques as per our published methods. Then biodistribution of exosomes carrying Ngb on the surface or in the lumen ±pFUS will be determined using In-111-tagged respective exosomes at the sites of stroke by SPECT-CT. We will also determine the distribution of exosomes with or without RVG peptides. Specific Aim 2: Investigate the recovery from a stroke following delivery of engineered exosomes carrying Ngb with or without pFUS. Exosomes will be delivered by pFUS after 24hrs of stroke; stroke recovery will be determined using MRI, and a battery of short- and long-term behavioral outcome tests will be performed followed by immunohistochemistry on brain tissue samples.